Method and system for automating sample preparation for microfluidic cryo TEM

ABSTRACT

A method and system is provided for automatically preparing transmission electron microscopy (TEM) samples for examination by depositing extremely small samples onto a grid without need for a blotting step. A sample liquid droplet is formed at the end of a capillary, wherein a portion of the liquid is transferred to the TEM sample grid by contact. The excess volume in the liquid droplet is then retracted by an adjacent capillary. After a predetermined time interval, the retraction capillary is moved toward the drop of the sample to remove the excess volume. As compared to a conventional machine, where the blotting procedure can deform the structure of the molecule of interest, the present invention utilizes a very low shear rate for removal of the excess sample fluid.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is related to and claims priority from earlier filedU.S. Provisional Patent Application No. 61/317,069, filed Mar. 24, 2010,the contents of which are incorporated herein by reference.

GOVERNMENT LICENSE RIGHTS

This invention was made with government support under CBET0730392awarded by National Science Foundation. The government has certainrights in the invention.

BACKGROUND OF THE INVENTION

The present invention relates generally to a method and system ofpreparing specimens for use in transmission electron microscopy (TEM).More specifically, the present invention relates to a method and systemfor automatically preparing TEM samples for examination by depositingextremely small samples onto a grid without need for a blotting step.

Transmission electron microscopy (TEM) is a microscopy technique wherebya beam of electrons is transmitted through an ultra-thin specimen suchthat the electron beam interacts with the specimen as it passes through.The interaction of the electrons transmitted through the specimen inturn creates an image. This created image is then magnified and focusedonto an imaging device, such as a fluorescent screen, a layer ofphotographic film, or is detected by a sensor such as a CCD camera. Thebenefit is that TEM systems are capable of imaging at a significantlyhigher resolution than traditional light microscopes due to the smallwavelength of the electron beams. This enables the instrument's user toexamine fine detail, such as a single column of atoms, which is tens ofthousands times smaller than the smallest resolvable object in a lightmicroscope. TEM forms a major analysis method in a range of scientificfields, in both physical and biological sciences.

TEM specimens must be prepared and placed into gridded specimen holdersto allow for insertion of the specimen holder into a vacuum column. Thesample is placed onto the inner meshed area of the grid. Usual gridmaterials are copper, molybdenum, gold or platinum. This grid is placedinto the sample holder which is paired with the specimen stage. A widevariety of designs of stages and holders exist, depending upon the typeof experiment being performed.

The principal difficulty in the prior art is that sample preparation inTEM can be a complex procedure. TEM specimens are required to be at mosthundreds of nanometers thick because the electron beam interacts readilywith the sample, an effect that increases roughly with atomic numbersquared. High quality samples will have a thickness that is comparableto the mean free path of the electrons that travel through the samples,which may be only a few tens of nanometers. Preparation of TEM specimensis specific to the material under analysis and the desired informationto obtain from the specimen. As such, many generic techniques have beenused for the preparation of the required thin sections.

Materials that have dimensions small enough to be electron transparent,such as powders or nanotubes, can be quickly prepared by the depositionof a dilute sample containing the specimen onto support grids or films.The difficulty is that the deposition of these solutions generallyresults in the creation of a droplet that is too large and thick forsampling requiring that the sample be blotted. This is accomplishedthrough the use of filter paper which introduces high shear to thespecimen and is done at the expense of a great deal of time, both ofwhich limit the ability to examine certain specimens and the formationof certain natural structures over time.

Once the specimen is deposited, in the biological sciences in order towithstand the instrument vacuum and facilitate handling, biologicalspecimens can be fixated using either a negative staining material suchas uranyl acetate or by plastic embedding. Alternately samples may beheld at liquid nitrogen temperatures after embedding in vitreous ice.The biological material is spread on an electron microscopy grid and ispreserved in a frozen-hydrated state by rapid freezing, usually inliquid ethane near liquid nitrogen temperature. By maintaining specimensat liquid nitrogen temperature or colder, they can be introduced intothe high-vacuum of the electron microscope column.

There is therefore a need for an automated system for the preparation ofa TEM sample. There is a further need for an automated system thatautomatically places and controls the size of a sample droplet formed ina TEM sample while eliminating the need for a blotting step. There is afurther need for an automated system that automatically places andcontrols the size of a sample droplet formed in a TEM sample whileeliminating the need for a blotting step in a manner that acceleratesthe preparation process thereby allowing previously unobservedstructures to be viewed.

BRIEF SUMMARY OF THE INVENTION

In this regard, the present invention provides a method and system ofpreparing specimens for use in transmission electron microscopy (TEM).More specifically, the present invention provides a method and systemfor automatically preparing TEM samples for examination by depositingextremely small samples onto a grid without need for a blotting step.

Most generally, the present invention provides a method and system forthe formation of a sample liquid droplet at the end of a capillary,wherein a portion of the liquid is transferred to the TEM sample grid bycontact. The excess volume in the liquid droplet is then retracted by anadjacent capillary. The flow rate of injection/retraction and the growthof the drop on the capillary tip are initially evaluated to tune thestarting time of the retraction capillary motion. After a predeterminedtime interval, the retraction capillary is moved toward the drop of thesample to remove the excess volume. As compared to a conventionalmachine, where the blotting procedure can deform the structure of themolecule of interest, the present invention utilizes a very low shearrate for removal of the excess sample fluid.

Once the sample is deposited and retracted the grid is then be plungedinto liquid ethane to vitrify the sample. By eliminating the blottingstep and controlling flow rates and residence times in the microchannels, the system of the present invention can facilitate the directvisualization of aggregates that are formed within the ten to hundredmillisecond time scales. All of the operation described here will becontrolled by a computer, allowing very accurate knowledge ofmicrostructure age at the time of freezing. This device will represent aunique window for the examination of the temporal evolution of aggregatemorphologies.

It is therefore an object of the present invention to provide anautomated system for the preparation of a TEM sample. It is a furtherobject of the present invention to provide an automated system thatautomatically places and controls the size of a sample droplet formed ina TEM sample while eliminating the need for a blotting step. It is stilla further object of the present invention to provide an automated systemthat automatically places and controls the size of a sample dropletformed in a TEM sample while eliminating the need for a blotting step ina manner that accelerates the preparation process thereby allowingpreviously unobserved structures to be viewed.

These together with other objects of the invention, along with variousfeatures of novelty which characterize the invention, are pointed outwith particularity in the claims annexed hereto and forming a part ofthis disclosure. For a better understanding of the invention, itsoperating advantages and the specific objects attained by its uses,reference should be had to the accompanying drawings and descriptivematter in which there is illustrated a preferred embodiment of theinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings which illustrate the best mode presently contemplatedfor carrying out the present invention:

FIG. 1 is an enlarged view of the fluid deposition system and method ofthe present invention wherein a fluid droplet is formed at the end ofthe deposition capillary;

FIG. 2 is an enlarged view of the fluid deposition system and method ofthe present invention wherein a fluid droplet is brought into contactwith the TEM sample grid by the deposition capillary;

FIG. 3 is an enlarged view of the fluid deposition system and method ofthe present invention wherein a fluid droplet remains on the TEM samplegrid as the deposition capillary is withdrawn;

FIG. 4 is an enlarged view of the fluid deposition system and method ofthe present invention wherein a retraction capillary is brought intocontact with the fluid droplet;

FIG. 5 is an enlarged view of the micro fluidic sample remaining on theTEM sample grid after the capillaries are withdrawn; and

FIG. 6 is a view of an overall system incorporating the fluid depositionsystem and method of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

Now referring to the drawings, the present invention can be seen toprovide a method and system of preparing transmission electronmicroscopy (TEM) samples for examination by depositing extremely smallsamples onto a grid without need for a blotting step. As best shown atFIG. 1, the present invention generally provides a method and system forthe formation of a sample liquid droplet 10 at the end of a capillary12, wherein a portion of the liquid 10 is transferred to the TEM samplegrid 14 by direct contact of the droplet 10 therewith. The excess volumein the liquid droplet is then retracted by an adjacent capillary 16. Theflow rate of injection/retraction and the growth of the drop on thecapillary tip are initially evaluated to tune the starting time of theretraction capillary motion. After a predetermined time interval, theretraction capillary is moved toward the drop of the sample to removethe excess volume. As compared to a conventional machine, where theblotting procedure can deform the structure of the molecule of interest,the present invention utilizes a very low shear rate for removal of theexcess sample fluid.

In connection with cryo-TEM processes, once the sample is deposited andretracted the sample grid is then be plunged into liquid ethane tovitrify the sample. By eliminating the blotting step and controllingflow rates and residence times in the micro channels, the system of thepresent invention can facilitate the direct visualization of aggregatesthat are formed within the ten to hundred millisecond time scales. Allof the operation described here will be controlled by a computer,allowing very accurate knowledge of microstructure age at the time offreezing. The range of applicability of the presentmicrofluidic-cryo-TEM device disclosed herein serves to (1) eliminatethe blotting step during sample preparation; (2) automate thepositioning of the capillary deposition device with the sample grid; and(3) integrate the microfluidic device inside the CEVS box.

As was stated previously, in sequential operation the method of thepresent invention is particularly illustrated at FIGS. 1-5. In operationas seen at FIG. 1, the depositing capillary 12 allows a flow thatcreated a fluid droplet 10 at the end thereof. In FIG. 2, the depositingcapillary 12 is brought into contact with the sample grid 14 leaving thesample droplet 10 thereon. At FIG. 3 the depositing capillary 12 iswithdrawn and the retracting capillary 16 is extended. At FIG. 4, whenthe retracting capillary 16 comes into contact with the droplet 10,excess fluid is drawn into the retracting capillary 16. As shown at FIG.5, both the depositing capillary 12 and the retracting capillary 16 havebeen withdrawn leaving a microfluidic sample 10 on the EM sample grid14. This process is then repeated automatically as necessary for anyadditional cells on the TEM sample grid 14.

The system operation is monitored and carefully controlled via computerso as to optimize the gap between grid 14 and retracting capillary 16and its velocity (time of contact) will be optimized. The flow rate ofinjection/retraction and the growth of the drop 10 on the depositingcapillary 12 tip are evaluated to tune the starting time of retractioncapillary 16 motion. After a predetermined time interval (this time isdictated by the required temporal resolution), the retraction capillary16 is moved as shown at FIGS. 3 and 4 toward the drop 10 of the sampleand remove the excess volume. The system employs an automated XYZpositioning system that positions the deposition capillary 12 andcontrols the contact time control between liquid 10 from depositioncapillary 12 to the cryo-TEM sample grid 14. An alignment camera mayalso be used for alignment feedback control. Once the meniscus of thedroplet 10 from the deposition capillary 12 touches the grid 14, asshown in FIG. 2, the retracting capillary 16 moves forward as shown atFIGS. 3 and 4 to grid 14 to remove the excess fluid 10.

Upon deposition onto the grid 14 the liquid ejecting from the depositioncapillary 12 should spread easily on the grid 14. Preferably the grid ismade hydrophilic to enhance the spread of the liquid samples. Byeliminating the blotting step and controlling flow rates and residencetimes in the micro channels of the capillaries it is expected thatdirect visualization of aggregates that are formed within the ten tohundred millisecond time scales can be achieved. All of the operationdescribed here will be controlled by a computer, allowing very accurateknowledge of microstructure age at the time of freezing. This devicewill represent a unique window for the examination of the temporalevolution of aggregate morphologies.

As can be seen at FIG. 6 the microfluidic device of the presentinvention may be incorporated into the CEVS box 18 controlled by acomputer 20. A judiciously designed microfluidic setup offers a uniqueopportunity to rapidly screen the short and long time scale formation ofnanomaterials, soft colloids and vesicles. The key feature that permitsthis is the laminar flow of the sample fluids in the capillary 12, 16channels, diffusive transport in a direction orthogonal to the flow, andthe small volumes that are processed allowing the temperature toequilibrate easily. The mixing of two solutions decreases as themagnitude of the flow increases relative to the diffusional flow acrossthe channel. The convective and diffusional transport of thesupramolecules can be described using a Peclet number. The Peclet numbercan be varied from below 1 to above 1 by manipulating the flow rates inthe channels. This allows control over extent of diffusive mixing in thechannel. The images will provide a fundamental basis to understandingthe effects of concentration, pre-shear and mixing time on structure andhomogeneity.

Using this new microfludic integrated cryo-TEM CEVS, the structureformation during precipitation/crystallization of inorganic and hybridsystems will be explored. Supplying the starting materials necessary forthe initiation of solid-state formation (be it via nucleation, spinodalcomposition or interfacial reactions) into the microfluidics deviceallows for the tracking of the reaction and structure formation at theinterface between the two laminar streams along the flow channel. Whentwo supersaturated solutions are mixed from their reservoirs, crystalformation initiates at the plane common to the two solutions in themicrofluidic channel. Structures formed there will be examined directlyby cryo-TEM using the computer-controlled setup developed. Since this isa flowing system, and crystals form only at the zone where there issufficient ‘contact’ between the two solutions, there is no danger ofplugging the microchannels. The concentrations, relative flow rates ofeach feed solution and the residence time in the channel will becontrolled to allow us to examine a very wide range of conditions quiteeasily. Our new TEM system, which allows low-dose capability, willpermit diffraction studies to be performed easily, so that thecrystalline nature of any of the precursor phases can be examined. Incrystalline phases, we will look closely for polymorphs.

Furthermore, the influence of water-soluble polymers (for examplepolycarboxylic acids) on the structural evolution will be studied. It isknown that polymers exhibit a range of modes-of-action, such ascomplexation, stabilization, flocculation. The role of additives will beexplored using such a system, by adding a reservoir containing theadditive and controlling the flow rate from this reservoir. Only bymeans of time-resolved experiments is it possible to unequivocally trackdown these mechanisms to their molecular origin.

Finally, it is possible to examine particle formation in organicsystems. This field is of great importance since half of the presentlysynthesized active pharmaceutical ingredients (API) are too hydrophobicto be applied in standard formulations. One approach to improve thebioavailability is too reduce the size of the primary API particles andthus to increase the solubility. Again, nanoparticle formation duringprecipitation can be studied with the unique approach—combiningmicrofluidics and cryo-TEM—as a well known hydrophobic active shall beused to study particle formation and the influence of stabilizingagents, such as copolymers.

It can therefore be seen that the present invention provides anautomated system for the preparation of a TEM sample that automaticallyplaces and controls the size of a sample droplet formed whileeliminating the need for a blotting step. Further, the present inventionprovides an automated system that automatically places and controls thesize of a sample droplet formed in a TEM sample while eliminating theneed for a blotting step in a manner that reduces shear induced in thesample and accelerates the preparation process, thereby allowingpreviously unobserved structures to be viewed. For these reasons, theinstant invention is believed to represent a significant advancement inthe art, which has substantial commercial merit.

While there is shown and described herein certain specific structureembodying the invention, it will be manifest to those skilled in the artthat various modifications and rearrangements of the parts may be madewithout departing from the spirit and scope of the underlying inventiveconcept and that the same is not limited to the particular forms hereinshown and described except insofar as indicated by the scope of theappended claims.

What is claimed:
 1. A method of preparing a cryogenic transmissionelectron microscopy sample grid, comprising: providing a sample carriergrid having nanoscale sample openings; forming a fluid droplet at theend of a first capillary; bringing said droplet into contact with a topsurface of said carrier grid thereby depositing said droplet onto saidtop surface; and bringing a second capillary onto contact with saiddroplet to withdraw a portion of the fluid from said droplet to form amicrofluidic sample on said top surface, wherein said steps of forming afluid droplet, bringing the droplet into contact with a top surface andbringing a second capillary into contact with the droplet are controlledusing a computer.
 2. The method of claim 1, further comprising: afterwithdrawing a portion of said fluid, freezing said carrier grid withsaid microfluidic sample thereon.
 3. The method of claim 1, wherein thecomputer controls an automated XYZ positioning system that positions thefirst capillary and controls a contact time between the fluid dropletand the top surface.
 4. The method of claim 1, wherein the top surfaceof said carrier grid is hydrophilic.
 5. A system for preparing acryogenic transmission electron microscopy sample grid, comprising: anautomatic controller; a first capillary for depositing fluid samplesonto a sample carrier grid having nanoscale sample openings; a secondcapillary for withdrawing a portion of said fluid samples; and anautomated XYZ positioning controller in communication with saidautomatic controller, said XYZ positioning controller controlling theposition of said first and second capillaries, wherein said automaticcontroller controls said first and second capillaries by, forming afluid droplet at the end of said first capillary; bringing said dropletinto contact with a top surface of a carrier grid thereby depositingsaid droplet onto said top surface; and bringing said second capillaryonto contact with said droplet to withdraw a portion of the fluid fromsaid droplet to form a microfluidic sample on said top surface.
 6. Thesystem of claim 5, further comprising: said automatic controllercontrolling freezing said carrier grid with said microfluidic samplethereon.
 7. The system of claim 5, wherein said automatic controllercontrols said automated XYZ positioning controller to position the firstcapillary and control a contact time between the fluid droplet and thetop surface.
 8. The system of claim 5, wherein the top surface of saidcarrier grid is hydrophilic.